Salicylic acid and jasmonic acid-mediated different fate of nickel phytoremediation in two populations of Alyssum inflatum Nyár.

This study investigates Ni phytoremediation and accumulation potential in the presence of salicylic acid (SA) (0, 50 and 200 μM) and jasmonic acid (JA) (0, 5 and 10 μM) in two populations of Alyssum inflatum under various nickel (Ni) doses (0, 100 and 400 μM). By measuring Ni levels in the shoots and roots, values of bioaccumulation coefficient (BAC), biological concentration factor (BCF) and translocation factor (TF) were calculated to quantify Ni accumulation and translocation between plant organs. Additionally, the amounts of histidine (His), citric acid (CA) and malic acid (MA) were explored. The results showed that plant dry weight (DW) [in shoot (29.8%, 8.74%) and in root (21.6%, 24.4%)] and chlorophyll [a (17.1%, 32.5%), b (10.1%, 30.9%)] declined in M and NM populations respectively, when exposed to Ni (400 μM). Conversely, the levels of MA [in shoot (37.0%, 32.0%) and in root (25.5%, 21.2%)], CA [in shoot (17.0%, 10.0%) and in root (47.9%, 37.2%)] and His [in shoot (by 1.59- and 1.34-fold) and in root (by 1.24- and 1.18-fold)] increased. Also, in the presence 400 μM Ni, the highest accumulation of Ni was observed in shoots of M (1392 μg/g DW) and NM (1382 μg/g DW). However, the application of SA and JA (especially in Ni 400 μM + SA 200 μM + JA 5 and 10 μM treatments) mitigated the harmful impact of Ni on physiological parameters. Also, a decreasing trend was observed in the contents of MA, CA, and His. The reduction of these compounds as important chelators of Ni caused a decrease in root-to-shoot Ni transfer and reducing accumulation in the shoots of both populations. The values of phytoremediation indices in both populations exposed to Ni (400 μM) were above one. In presence of the SA and JA, these indices showed a decreasing trend, although the values remained above one (BAC, BCF and TF > 1). Overall, the results indicated that SA and JA can reduce phytoremediation potential of the two populations through different mechanisms.

limiting its absorption and entry into the root cells 31 .The current study focused on investigating the patterns of Ni accumulation and transport in metallicolous (M) and non-metallicolous (NM) populations of A. inflatum under the treatment of SA and JA.Therefore, SA and JA were used as regulatory factors to explore their role in reducing the effects of Ni toxicity and phytoremediation in the two populations of A. inflatum.The aim was to provide a clear understanding of the modulatory impacts of these phytohormones and their interaction on Ni stress in plants, as well as on the phytoremediation of contaminated soils by Ni-accumulating plants.

Plant preparation and treatments
Seeds of Alyssum inflatum Nyár. of the M population were collected from serpentine sites in Marivan (35 ° 13.625′ N; 46 ° 27.184′ E), and the seeds of NM populations were gathered from non-serpentine of Shahu sites (34 ° 56′ 47′′ N; 46 ° 27′ 41′′ E), respectively from West of Iran in summer of 2015 10 by Naser Karimi (Department of Biology, Razi University, Kermanshah, Iran) according to the institutional, national and international guidelines and laws.The permission to collect plants (license code: IR.KUMS.REC.1401.512)was obtained from the Pharmaceutical Sciences Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran, which identifies the institutional licensing committee that approves the collection of plants and experiments, including any relevant details.The plant populations were identified after being collected and transferred to the laboratory by Hosein Maroofi (Agricultural and Natural Resources Research and Education Center, Kurdistan, Iran).A voucher specimen of the plant was deposited in the dedicated herbarium of the Kurdistan Agricultural and Natural Resources Research and Education Center (No. 9562-HKS), which is available to the public upon request.All seeds were kept at 4 °C.To start the experiments, the surface of the seeds was first sterilized with 1% (v/v) sodium hypochlorite solution (NaOCl) for 10 min, then thoroughly washed with distilled water.They were planted in 162 pots with a volume of 450 mL [contained perlite: sand (2:1) mix], and each pot was placed in a bucket.A total of 81 pots were assigned to each population, and eight seeds from each population were planted in each pot.The seeds were watered with tap water for 15 days until germination, after which they were fed with a modified Hoagland solution according to the instructions of Najafi-Kakavand et al. (2019) 11 .Also, Hoagland's solution for entire pots was replaced every 5-days.All pots were kept in the controlled conditions in a CG 72 Environ growth chamber [light (16 h, 25 °C)/dark (8 h, 16 °C) period, PPFD of 140 μmol m −2 s −1 ].After 45 days of germination, all pots of each population were distributed in 27 treatment groups, and three pots were given to each treatment group.Applied treatments included different doses of Ni (0, 100 and 400 μM), JA (0, 5 and 10 μM), and SA (0, 50 and 200 μM) individually or in combination.Every 7 days, the treatments were applied alone or combined with Ni and SA (along with nutrient solution) and JA (by spraying the leaves) for 21 days.After the treatment period, all the A. inflatum plants in both populations were harvested and divided into the root and shoot segments for more investigation and held at − 70 °C.

Dry weight measurement
At harvesting (21 days after exposure), root and shoot DW were measured.For this purpose, the separate parts of the shoot and root of each plant (three repetitions per treatment) were dehydrated in an oven at 75 °C for 72 h till stable weight.

Chlorophyll content measurement
Extraction of photosynthetic pigments from leaf samples of two populations of A. inflatum was performed using Arnon (1949) method 32 .First, 100 mg of fresh leaf material was homogenized in 10 mL of cold 80% acetone, then centrifuged for 15 min (at 4000 rpm, 4 °C).The absorbance of chlorophyll a and b were assayed by spectrophotometer at A 663 and A 645 nm, respectively.Finally, the amount of photosynthetic pigment was estimated with the equations mentioned below:

Ni content assayed
Ni levels in two parts (root and shoot) of both populations of A. inflatum were determined based on the method introduced by Ghasemi et al. (2009) 33 .Briefly, dried matter samples were digested with 60% nitric acid (2 mL) for 24 h at room temperature, followed by incubating at 90 °C.After 4 h of heating, the samples were cooled.Then, by adding 1 mL of H 2 O 2 , the test tubes containing the samples were again exposed to 90 °C in the water bath until the solutions were clarified.Finally, by adding deionized water, the final volume of each sample reached 10 mL.Ni analysis of shoots and roots was accomplished using atomic absorption spectrophotometry (AAS, Shimadzu model 6200).

Phytoremediation potential
To assess Ni phytoremediation potential in two populations of A. inflatum, three parameters were used including: Biological concentration factor (BCF) was estimated as the ratio of Ni contents (μg/g DW) in root tissue (C root ) to Ni levels (μM) in roots' environment solution (C solution ) 34 , which was calculated as follows: www.nature.com/scientificreports/Bioaccumulation coefficient (BAC) was described as a ratio of Ni (μg/g DW) in the aerial part of a plant to the Ni contents (μM) in the roots' environment solution 35 , which was calculated as follows: A higher ratio points to better phytoaccumulation efficiency 36 .Translocation factor (TF) was evaluated as the ratio between Ni concentrations (μg/g) in aerial parts (C shoot ) to Ni concentration (μg/g) in roots (C root ), which was calculated as follows: TF > 1 indicates that A. inflatum plants transfer Ni effectively from the ground parts to aerial parts 37 .

HPLC analysis
Preparation of plant extract: Using the ultra-sonication-assisted extraction (UAE) technique defined by Krishna  et al. (2018), the plant extracts were prepared 38 .First, 5 mL of deionized water was added to 100 mg of dry-matter powder and homogenized by a sonication bath for 30 min.Homogenous samples were centrifuged (3000 rpm, 15 min) to obtain a clear plant extract.The supernatant was held in a cold place (refrigerator) and analyzed within 12 h of preparation.
HPLC method: The evaluation of citric acid (CA), malic acid (MA) and histidine (His) contents was performed by an isocratic mode at 1 mL/min flow rate with a Knauer HPLC instrument (equipped with Smartline 1000 quaternary pump version 7603, UV-detector 2600 version 7605, and Chrom Gate HPLC software 3.1.7)on an Eurospher 100-5 C18 column (250 × 4.6 mm, 5 μm) in the room temperature.The solvent system contained buffer phosphate (100 mM; pH 2.5).The samples' analysis was recorded at 210 nm.The volume of the injection for each sample was 40 μL.Three replicates were prepared from each sample.The chromatographic peaks of CA, MA and His in plant samples were certified by comparison of their retention time and UV-spectrum with those of the corresponding standards of referral to the standard.Measurements were made based on the linear calibration curves obtained from the standard solutions of CA and MA and His and the area under the curve of peaks of the standards and the plant extracts.

Data analysis
A randomized complete block design (RCBD) with three replications was used to conduct a factorial experiment involving 4 factors to analyze variance.Factors in this experiment included different doses of Ni, SA and JA (mentioned in part 2.1) along with two distinct populations of A. inflatum seeds (M and NM).The LSMEANS statement (SAS ver.9.4) was applied when the interaction between treatments was significant.All the values are presented in the tables and figures as mean ± SE (standard error).Principal component analysis (PCA) was performed using the fviz-pca function of the factoextra R package ver.1.0.7 39 to visually biplot treatments and variables.

Shoot and root dry weight
As an essential element, plants need low quantities of Ni for optimal growth and development.However, higher concentrations of Ni cause toxicity and can lead to several harmful changes in plant physiology and anatomy 40 .In the present study, excess Ni doses can negatively affect plant growth; therefore, with increasing Ni concentration, a significant decrease was detected in the shoot and root DW of both populations of A. inflatum.The highest reduction in the shoot DW with 29.8% of M population and 21.6% and 24.4% in roots DW were observed in M and NM populations exposed to 400 μM Ni, respectively, compared to control (Table 1).One of the causes of reduced plant growth and biomass production in the presence of high concentrations of Ni is the lack of essential elements such as iron (Fe), copper (Cu), and manganese (Mn).Due to similar chemical properties, Ni competes with these elements for absorption through their transporters in the root 10 .Accordingly, Rathor et al. (2014) demonstrated a significant decline in dry matter yield in Zea mays plants due to the accumulation of elevated doses of Ni 41 .Another study showed that Ni at lower concentrations (0-5 mg Ni/L) induced a meaningful increment in dry matter yield, but at higher concentrations (6-25 mg/L) caused toxic effects and led to a significant decrease in dry matter yield 42 .However, the reduction in biomass of plants exposed to Ni stress is related to plant metabolism, photosynthesis, water relations, transpiration, disturbance in the absorption of the nutrient, and oxidative damage caused by Ni stress 43 .Based on Table 1, using SA and JA alone or in combination in Nistressed plants in both populations exhibited different effects on plant mass.Using of SA augmented shoot and root dry matter of M plants, as well as root DW of NM plants under Ni treatments.In contrast, JA decreased the shoot DW of both populations under Ni treatments.The SA plays a main role in the regulating of cell growth, seed germination, seedling development, and ion uptake and transport.JA influences root growth by preventing the primary root, forming of lateral roots, regenerating the root, and reducing adventitious root formation 23 .As shown in Table 1, the application of SA in high doses (200 μM) caused the highest DW in shoots (30.3%) and roots (27.5%) of M population plants under the highest Ni concentration relative to plants under 400 μM Ni stress alone.A similar result in the same treatment was displayed in the root region of the NM population (46.03%).Similarly, in strawberry plants, applying Ni (150-300 mg/L) with SA (2 mM) increased the DW and FW of shoots and roots 44 .Unlike SA, JA in the presence of high Ni concentrations did not induce significant alterations in shoot biomass of both populations.In contrast to this result, Azeem (2018) indicated that in Z. mays plants, JA at 6-10 μM increased the seedling emergence, leaf number, and shoot length alone and in combination with Ni (8 μM) 45 .According to this study, JA via elevating the action of antioxidant enzymes ameliorated BAC = C shoot /C solution TF = C shoot / C root the damaging impacts of oxidative stress on biomass production, growth, and protein amounts in Ni-stressed plants.However, JA treatment increased root biomass in plants against 400 μM Ni stress in both populations to plants against the same dose of Ni.The highest DW of the M population was detected in the presence of 400 μM Ni + 50 and 200 μM SA + 10 μM JA.In this condition, the shoots and roots DW were 1.16-1.23 and 1.13-1.31times, respectively, more than the stressed plants under 400 μM Ni alone.However, an enhancement in biomass by simultaneous application of SA with JA was observed only in the roots of the NM population under high Ni toxicity.For example, the highest increase in root DW (40.4%) was detected in 400 μM Ni + 200 μM SA + 5 μM JA treatments concerning the stressed plants under 400 μM Ni alone.Based on Table 1, the maximum ratio R/S was detected in plants exposed to 400 μM Ni + 10 μM JA by 1.49-and 1.84-fold increment, in M and NM populations respectively, compared to untreated plants.In the same situation, the highest enhancement of this ratio was detected in the treatment of 400 μM Ni + 200 μM SA only in the NM population (1.84).Also, using SA and JA externally in plants against high Ni concentration only boosted the R/S ratio in the NM population by 1.54 compared to the control plant.Therefore, the positive impacts of SA and JA in improving the growth parameters of both populations of A. inflatum can be related to their role in the internal changes of phytohormone levels such as abscisic acid (ABA) in regulating the function of stomata and transpiration, enhancing the biosynthesis of photosynthetic pigments and ameliorating the antioxidant system 11 .Similarly, Zaid et al. stated that biomass of Brassica juncea L. decreased significantly in the presence of Ni stress particularly at 150 μM, relative to the control group.Still, co-application of Ni with SA augmented shoot and root biomass 28 .It has been reported that the exogenous application of JA at 0, 1, and 60 μM doses in sunflower seedlings growth media decreased the primary root development and reduced the number of lateral roots 46 .However, JA and SA act as essential signaling agents to adjust the defensive response in plants against abiotic stress, which can be due to the nature, exposure time, and intensity of stress in plants 23 .

Photosynthetic pigments contents
The destructive effect of Ni toxicity on physiological processes, including photosynthetic pigments, has been proven.As expected, augmenting Ni level led to a declining tendency in chlorophyll a and b content in both studied populations of A. inflatum.The Ni toxicity (at 400 μM) led to a decline in chlorophyll a concentration by 17.1% and 32.5%, and chlorophyll b content by 10.1% and 30.9% in the M and NM plants of A. inflatum, respectively, in comparison with the untreated plants (Fig. 1).Excessive amounts of Ni might directly destroy the photosynthetic machine of leaves via different routes.Excessive Ni can smash epidermal and mesophyll cells, disrupt thylakoid membranes and grana structures of chloroplasts, reduce grana size, and raise the number of lamellae in stressed areas.These alterations diminish chlorophylls, carotenoids, and xanthophylls amounts 14,47 .In fact, oxidative stress induced by elevating doses of Ni decreases the electron transport in the photosynthetic electron transport chain, a disorder in the activity of reaction centers (P680, and P700), and delays chlorophyll synthesis 48 .
In line with this study, Ni at concentrations of 50 µM and 100 µM meaningfully declined the chlorophyll pigments (a, b and total) in cotton plants relative to the untreated plants 49 .Also, chlorophyll a and b contents in Z. mays plant leaves decreased after 13 days of plant growth under 100 and 200 mM Ni concentrations 50 .Similar to our results, Srivastava et al. showed that Ni considerably reduced the chlorophyll a/b ratio at concentrations of 0.1 and 1 mM, indicating that chlorophyll a was greater sensitive to Ni than chlorophyll b 51 .
The presence of SA or JA in both populations' growth media, especially at higher doses, led to a drop in the amount of chlorophyll a and b compared to control group.In this investigation, different impacts of SA and JA on chlorophyll content were evident in two A. inflatum populations against Ni toxicity.In both populations exposed to 400 μM Ni, SA, especially at high concentration (200 μM), caused a slight increase in chlorophyll content with respect to plants exposed to 400 μM Ni only (Fig. 1).One of the critical roles of SA is the regulation of photosynthetic pigments (chlorophyll and carotenoid), photosystem II (PSII) and the performance of carbonic anhydrase and Rubisco enzymes against HMs stress conditions 52 .It was also found that the co-treatment of SA and NO reduced Ni toxicity by improving photosynthetic apparatus, antioxidant enzymes' action, maintaining www.nature.com/scientificreports/nutrients homeostasis and reducing MDA formation 53 .However, JA, especially at high concentration (10 μM) in M plants exposed to 400 μM Ni, showed a reducing trend of chlorophyll content.In contrast, the NM population treated with SA or JA alone with 400 μM Ni resulted in an augmenting trend of chlorophyll content compared to plants under Ni stress alone (Fig. 1).External application of JA improved the chlorophyll amount and development of Glycine max seedlings exposed to Ni stress 48 .Overall, the highest amounts of chlorophyll a and b in both populations were attended in the treatment of 400 µM Ni + 200 µM SA + 5 µM JA, which led to an enhancement in the amount of chlorophyll a (of 1.69-and 1.88 times) and chlorophyll b (of 1.49-and 1.06 times) in M and NM populations, respectively, compared to the plants treated with Ni (400 µM) alone (Fig. 1).Conferring to our results, the use of high doses of SA and JA leads to an increase in the chlorophyll a and b contents in both populations, which reduces the adverse impacts of Ni toxicity on the photosynthetic system by stimulating biosynthesis of photosynthetic pigments, reducing reactive oxygen species (ROS), and improving antioxidant system 11 .Accordingly, Kamran et al. reported that high doses of external JA alone inhibited the growth and photosynthesis rate in choy sum plants.Whereas, JA (5, 10, and 20 µM) mitigated chromium (Cr) toxicity (150 and 300 µM) and improved gas exchange and chlorophyll contents with respect to the Cr-alone treatment plants.In this study, the useful impact of JA was attributed to the protective roles of JA on the performance of the photosynthetic machine, biosynthesis of chlorophyll, guiding of stomata, and transport rates of CO 2 .These authors stated that reducing in Cr amounts in plant cells could be a significant reason for JA-induced stimuli on photosynthesis 31 28 , which confirmed the obtained results in the current work.In another study, the amount of photosynthetic pigments was significantly decreased in lead (Pb)-stressed maize plants as compared to non-exposed plants.Meanwhile, the exogenous usage of JA, SA, proline alone, and co-treatment with Pb significantly enhanced these pigment content in Pb-stressed and control maize plants.This was attributed to the protecting task of JA in enhancing the photosynthetic apparatus and uptake of key minerals against stress to Pb and the role of SA in falling chlorophyll disruption and MDA increase and electrolyte leakage in treated plants 54 .

Plant Ni concentrations
To survey the impact of SA and JA on Ni uptake and potential phytoremediation, we determined the amount of Ni accumulated in the shoots and roots of Ni-exposed M and NM populations.As shown in Tables 2 and 3, with increasing doses of Ni in the plant root environment, the amount of Ni uptake by the plant roots in both populations showed a considerable increase.Thus, at 400 μM Ni treatment, the Ni doses in the ground part of the M and NM populations were 832 and 886 μg/g DW, respectively, which was 16.3-and 25.6 times higher than ground part of control plants.The uptake of Ni by ground part of plants occurred through two principal mechanisms, active transport system and passive diffusion pathway.Different factors such as plant species, Ni form, Ni amount in the rhizosphere, soil pH, organic matters in the soil, and plant metabolism affect Ni transport 40 .Furthermore, the uptake of Ni ions can be regulated by amino acids because these compounds act as chelators that form complexes with metal ions such as Ni, thus enhancing Ni uptake in plants 55 .The uptake of divalent cations in roots usually happens through apoplastic binding and symplastic uptake systems 56 .One study reported that the ratio of apoplastic to symplastic absorption in Ni-hyperaccumulator and non-hyperaccumulator plants was 85-95% to 5-15%, indicating that the symplastic route is very restricted for both plants 57 .Due to the rise in Ni uptake by the plant's roots, the rate of Ni transport from the root-to-shoot of these plants enhanced, therefore, excessive Ni accumulation was detected in the aerial parts of M and NM populations.After uptake, Ni is transferred simply into the xylem and then guided to aerial parts of plants 58 , then in there, Ni is redistributed through the phloem to stem, leaves, and other parts of plants 40  Likewise, they confirmed that SA is important in controlling signaling molecules in developing and mediating plants' response to HM stress 60 .
As shown in Tables 2 and 3, external usage of SA in combination with JA or alone decreased Ni uptake in the shoot of Ni-stressed M and NM populations.In contrast, exogenous JA led to an increase in Ni absorption in the stressed plant shoot.The maximum reduction of Ni uptake in the aerial parts of M and NM populations was detected in the groups of 400 μM Ni + 200 μM SA (38.7% and 50.1%), and 400 μM Ni + 10 μM JA (16.5% and 35.6%), respectively, compared to plants treated with 400 μM Ni.Besides, a sharp reduction in Ni accumulation in the shoots of both populations was perceived with increasing doses of Ni under the effect of simultaneous treatment with SA and JA.Accordingly, the lowest shoot Ni accumulation was related to the 400 μM Ni + 50 μM SA + 5 μM JA in the stressed-M plants (with 56.8%) and 400 μM Ni + 200 μM SA + 10 μM JA in the stressed-NM plants (with 75%).According to our results, SA and JA, especially in high doses, led to a decrease in Ni absorption by the roots and a decrease in its accumulation in the shoots of both populations by a possible reduction in the expression of transporter genes involved in its uptake and translocation from root to shoot 11,61 .Accordingly, Ali et al. (2018) explained that amount of Cd in the aerial parts of Brassica napus increased in Cd stress conditions, but exogenous application of JA significantly decreased Cd accumulation in stressed plants.This study revealed that reduction in HMs absorption in reaction to external usage of stress phytohormones such JA, SA, abscisic acid (ABA), and brassinolide could be attributed to accumulation/ exudation of organic compounds 59 .Also, in another study, the mitigation of Cd ions uptake and protection of plants against HMs stress were attributed to the endogenous and exogenous SA in plants.SA pre-soaking inhibited the accumulation of Cd in the shoot as a result of a considerable reduction in roots BCF and Cd transport, which were calculated using the TF and BAC in shoots, respectively 62 .Nevertheless, the findings of the current study showed the role of SA and JA in Ni absorption and accumulation in stressed plants, which may indicate the formation of Ni-organic or amino acids complexes to the chelation and sequestration of Ni in the root and thus tolerance of plants against Ni toxicity.

Phytoremediation potential
Phytoremediation is an internal mechanism for the absorption of HMs and deposition in their tissues, which allows the removal of HMs from contaminated sites at a higher rate and subsequent harvesting at a lower cost 63 .Generally, three main factors TF (shoot-to-root ratio of HMs), BCF (root-to-soil ratio of HMs), and BAC (shootto-soil ratio of HMs) are used to estimate the phytoremediation potential.Plants are expected to be appropriate for phytoremediation if the values of these factors are higher than one (TF > 1, BCF > 1, and BAC > 1) 8,35 .The averages of TF, BCF, and BAC values in different parts of M and NM populations of A. inflatum were shown 41.4 ± 5.04 s 0 ± 0 q 0 ± 0 t 0.517 ± 0.015 q-t 10 23.5 ± 1.55 xy 54.4 ± 3.72 s 0 ± 0 q 0 ± 0 t 0.432 ± 0.002 q-v 200 0 23.5 ± 1.55 xy 54.4 ± 3.72 s 0 ± 0 q 0 ± 0 t 0.432 ± 0.002 q-v 5 38.6 ± 0.87 xy 60.1 ± 1.44 s 0 ± 0 q 0 ± 0 t 0.643 ± 0.03 l-p 10 31.5 ± 1.10 xy 55.5 ± 2.22 s 0 ± 0 q 0 ± 0 t 0.57 ± 0.04  65 .Overall, the accumulation of HMs in plants relies on the outer environment and inner physiological characteristics, including HMs bioavailability, physicochemical characteristics of soil, biodiversity of microbes in soil, root exudation, temperature, and the existence of transporters 66 .
According to Tables 2 and 3, external application of SA and JA alone or in combination in plants against various concentrations of Ni, with rising Ni doses, a reduction trend was observed in the BCF and BAC indices of both populations of A. inflatum; however, BCF and BAC values of these treatments were greater than one.As an exception, exogenous JA use, especially at a lower concentration (5 μM) in plants under Ni (100 μM) stress, led to an enhancement in the BAC value of the M population (6.36) by 1.7 times and the NM population (7.518) 0 ± 0 q 0 ± 0 t 0.568 ± 0.134 n-t 5 56.5 ± 13.9 xy 46.1 ± 4.07 s 0 ± 0 q 0 ± 0 t 1.226 ± 0.283 c-e 10 64.8 ± 15.0 w-y 50.8 ± 5.69 s 0 ± 0 q 0 ± 0 t 1.31 ± 0.451 c 50 0 31.9 ± 5.50 xy 30.8 ± 1.18 s 0 ± 0 q 0 ± 0 t 1.036 ± 0.181 e-h 5 16.9 ± 0.50 xy 30.8 ± 1.18 s 0 ± 0 q 0 ± 0 t 0.549 ± 0.028 n-t 10 15.1 ± 4.90 y 36.5 ± 4.036 s 0 ± 0 q 0 ± 0 t 0.413 ± 0.126 q-w 200 0 15.1 ± 4.90 y 36.5 ± 4.04 s 0 ± 0 q 0 ± 0 t 0.413 ± 0.126 q-w 5 40.4 ± 0.60 xy 53.5 ± 1.80 s 0 ± 0 q 0 ± 0 t 0.756 ± 0.026  (3.456), that showed this value was less than one (BAC < 1).Besides, the TF values incremented in the presence of JA, especially at the higher dose (10 μM) in 100 μM Ni-treated plants in both populations (TF > 1), whereas at the same dose of Ni, the existence of SA led to a discount in the two populations (TF < 1).Generally They suggested that SA probably prevents the accumulation of Ni in the aerial parts by reducing the root uptake and also reducing root-to-shoot Ni translocation 67 .Likewise, it was found that the callus cultures of two wheat genotypes, tetraploid (Durum-97) and hexaploid (Shafaq-06), which were under Cd toxicity, raised Cd accumulation in the callus with the augmentation of Cd doses in the medium.The usage of 0.5 mM SA led to decreased BCF value to less than 1 in higher concentrations of Cd 68 .Similarly, seed priming of Linum usitatissimum L. with SA reduced the amount of Cd in the root and shoot of this plant, and as a result, the values of BCF, BAC, and TF were reduced to less than one.In this way, SA prevented the toxicity of Cd in the plant by preventing the absorption of Cd and its transport to the shoot by xylem 62 .As confirmed by our results, Coelho et al. (2020) found that the use of JA resulted in the accumulation of arsenic (As) above 1000 μg/kg DW and enhanced BCF value (281) in Lemna valdiviana.They stated that JA leads to an enhancement in As accumulation in plants by modulating ROS homeostasis and signaling and promoting the antioxidant system 69 .On the contrary, the decreasing trend of Cd accumulation with significantly decreased BCF and TF values was observed in chickpea (Cicer arietinum) plants treated with JA and gibberellic acid (GA3) alone and/or in combination 70 .
It has also been proven that exogenously JA treatment can disrupt the absorption, transport (root-to-shoot), and accumulation of HMs by suppressing the transporter genes involved in the absorption of HMs by root from soil such as As (Lsi1, Lsi2, and Lsi6 genes) and Cd (AtHMA4 and AtHMA2 genes), as well as transporter genes related to HMs xylem loading such as Cd (AtIRT1 gene), in plants 71 .As indicated by our results, in the existence of SA and JA, the BCF value had an increasing trend compared to the BCF of plants exposed to 400 µM Ni alone in plants under high Ni concentration.In contrast, the values of BAC and TF exhibited a decreasing trend.Accordingly, SA and JA can probably prevent the accumulation of Ni in the aerial parts of plant by stimulating the uptake, sequestration, and precipitation of Ni in special organelles of root cells, such as vacuoles 72 , as well as disrupting the mechanisms of Ni translocation from root to shoot.

Citric acid (CA) and malic acid (MA) concentrations
Organic acids (OA) have multiple roles in plants; for example, MA, CA, and oxalate induce HMs tolerance by moving them via the xylem and sequestrating them in vacuoles 73 .According to Kocaman (2023), increasing the content of malonic acid and MA (up to 1 unit) in the plants can lead to binding (chelation) of 0.7 and 0.5 units of Ni 55 .In addition, OAs are chelated by their carboxyl groups with cationic HMs, thus reducing the amount of free toxic active ions.After entering, cationic HMs may stimulate OA channels or interact with a receptor protein and induce effective genes in the synthesis of OA.Occasionally, in the root surface, OA-HM complexes can arrive at cells through diffusion or by active transport via particular ligand/transporter channels and after that may sequester or accumulate in intracellular parts such as the vacuoles 74 .The content of OA including MA and CA as Ni chelators that role in its transfer and as well as stored in roots and shoots of M and NM populations of A. inflatum are displayed in Figs. 2 and 3. Interestingly, the CA and MA amounts in both populations' plant roots and shoots were raised due to an increment in Ni concentration in the plant growth medium.According to the results obtained from this experiment, in plant roots exposed to 400 μM Ni, the CA content was increased by 47.9% and 37.2% (Fig. 2), and as well as the MA content was augmented by 25.5% and 21.2% (Fig. 3), in the M and NM populations, respectively, in comparison to the untreated plant groups.As it is known, CA is synthesized in plants by citrate synthase enzyme.Compared to MA and oxalate, this OA has a greater tendency for metal ions such as Ni 2+ and Cd 2+ , although, its chief role is to chelate Fe 2+73 .Furthermore, in the shoot regions of plants under 400 μM Ni stress, the content of CA and MA augmented by 17.0% and 37.0% in the M population and by 10.0% and 32.0% in the NM population, respectively, relative to the unexposed plants group.Similarly, Amari et    www.nature.com/scientificreports/ the expression of enzyme genes related to the metabolism of OAs in tomato seedlings under lead stress under toxic doses of lead, accordingly the amount of CA synthase and MA synthase enzymes decreased.Still, seedlings exposed to Pb (0.75 mM) with JA (100 nM) detected an up-regulation of the expression of fumarate hydratase and succinate dehydrogenase enzymes 79 .In line with our findings, Kohli (2018) reported increased CA, MA, succinic acid, and fumaric acid contents in B. juncea under stress to Pb (0.75 mM).In this plant, the presence of SA (1 mM) and 24-epibrassinolide acid (EBL) (10 -7 M) alone led to a greater rise in the amounts of these OAs, as well as the simultaneous treatment with EBL + SA showed the greater effect than these treatments alone in increasing the amounts of these compound in Pb-treated seedlings.For example, the authors demonstrated 123% and 141% increases in CA and MA contents, respectively, under treatment with Pb + EBL + SA in stressed plants 80 .However, OAs such as CA and MA, similar to amino acids, have a role in Ni uptake through the formation of Ni-OA complexes by the roots and also cooperate with processes of root-to-shoot translocation and Ni precipitate in cell organelles such as vacuoles 40,72 .

Histidine (His) concentration
Different amino acids such as His, alanine, asparagine, proline, arginine, methionine, glycine, serine, cysteine, lysine, and glutamic acid, are known to accumulate at high doses against HMs (Ni, Pb, Cr, Zn, Cd) stress to preserve plants from the toxic impacts of certain metals 81 .To protect and tolerate the stressed plants, amino acids can act as compatible osmolytes, pH regulators and ROS detoxifiers as well as acting as nitrogen or carbon resources for the synthesis of certain enzymes and precursors of various secondary metabolites for instance flavonoids and lignin 55 .For example, His has been involved in the detoxifying of HMs in Ni-tolerant plants by its chelation 82 .Therefore, in the current study, the His levels related to the roots and shoots of both populations of A. inflatum were analyzed, as shown in Fig. 4. As be shown, high Ni doses enhanced the content of His in stressed plants.Its content was enhanced by 1.24-and 1.18-fold in the roots and incriminated by 1.59-and 1.34-fold in the shoots of the M and NM plants, respectively, with respect to the unstressed plants.It has been established that His has a high tendency to link with metals.Likewise, the free amino acids and proteins with (metal's coordination residues) have a partly high correlation constant (Log K: 8.7) for Ni 83  According to Fig. 3, the content of His in the shoots of two populations of A. inflatum under 400 μM Ni stress, in the presence of high doses of SA [in M population (14.0%),NM population (16.0%)] and JA [in M population (7.30%), NM population (24.5%)], exhibited a significant reduction over the plants stressed with Ni at a high dose.However, contradictory results were obtained in the root part of these plants.Besides, the simultaneous effect of SA and JA in combination at different concentrations in Ni-stressed plants caused a decrease in His content relative to the plants stressed with Ni alone.Although, compared to the unstressed plants, treatments of 400 μM Ni + 50 μM SA + 5 μM JA in M species roots by 1.13-fold and the treated group of 400 μM Ni + 200 μM SA + 10 μM JA in shoots of the M (1.36-fold) and NM (1.13-fold) populations and as well as the root of NM population by 1.2-fold caused an increase in the content of His.Considering that in the presence of SA and JA, the reduction of Ni accumulation in the shoots of the two populations under Ni stress occurred simultaneously with the reduction of His content.These results show the probable role of SA and JA in modulating Ni stress by reducing the expression of genes related to the biosynthesis of Ni chelators (such as His and OAs) and transporters involved in its uptake and transfer from root to shoot 83 .Similarly, the results obtained by Ahmad et al. (2021) indicated increasing proline and glycine betaine (GB) accumulation in chickpea plants under Cd stress supplemented with JA and GA 3 .They reported that the combination of JA and GA 3 in stressed plants (Cd + JA + GA 3 ) remarkably augmented proline and GB content by 6.08 times and 7.64 times increase, respectively, compared to control plants 70 .Furthermore, similar to our results, Bali et al. (2019) revealed that the treatment of tomato seedlings with 100 nM JA + 0.75 mM Pb reduced levels of His, cysteine, aspartic acid, isoleucine, leucine, and GABA (non-protein amino acid), while increased the content of other amino acids compared to plant stressed with 0.75 mM Pb alone.They pointed to the amino acids' role in nitrogen (N) metabolism that regulates the protein metabolism, re-mobilization of N, discount of nitrate and uptake of ammonium and nitrate 79 .Likewise, Zanganeh et al. (2019) reported that the usage of SA on the seeds of the Z. mays plant, as a pre-treatment, led to a reduction in the amount of His in plant shoots and roots under Pb treatment, while the application of sodium hydrosulfide (NaHS) on the plant seeds caused a decline in the content of roots His and an augmentation in the level of His in shoots.However, according to the authors' results, SA accumulated other amino acids similar to alanine, tyrosine, valine, tryptophan, leucine, and isoleucine.They explained that the decrease of amino acid levels by pre-treatment with SA and NaHS could be considered as a contribution to the beginning of adaptive processes with the unfavorable impact of Pb.Amino acids play precursor roles in synthesizing stress-related proteins and defensive metabolites against plant stress.On the other hand in their study, the increased content of His was attributed to the production of de novo and N storage to inhibit ammonium toxicity and as a result the increase in the contents of His and arginine amino acids, which are appropriate for N storage owing to the existence of binary amides in their structure 78 .These results were in line with our results, which observed that SA declined His levels in the shoots of both populations and root of the M population under Ni stress.However, the using exogenous phytohormones such as JA and SA can cause an increment in the content of endogenous amino acids in plants, resulting in improved plant tolerance to abiotic stress like HMs.This is achieved by enhancing plant metabolism through the regulation of membrane permeability, the activation of enzymatic process, the production of osmolytes, and the uptake of some ions 87 .

Principal component analysis (PCA)
Using PCA we investigate the interrelationships within a set of variables and elucidated them via diverse biplots.In our investigation of the correlation between variables and traits across two distinct populations of A. inflatum, we employed PCA independently for each population and collectively across both populations (Figs. 5, 6, 7).The first two principal components of PCA accounted for (57.7%), (57.6%) and (61.3%) of the variance when analyzing both populations collectively or when examining populations M and MN separately.Furthermore, PCA plots demonstrated a similar trend in both M and NM plants in response to the presence of SA and JA in plants under Ni stress.The PCA results revealed that in the M (Fig. 5) and NM (Fig. 6) populations, in the presence of SA and JA supplements under Ni toxicity, there was an approximate positive correlation between plant biomass, amount of Ni and the levels of His, MA, SA, and phytoremediation indices (TF, BAC, BCF).This pattern was also seen in the common PCA plot between the two populations (Fig. 7).Interestingly, the highest correlation between phytoremediation parameters and Ni levels of roots and shoots was observed in population M. The pattern was also observed in the NM population.On the other hand, the lowest correlation value between chlorophyll (a, b) levels and phytoremediation parameters was detectable in all PCA plots.In total, the findings of this study and

Conclusion
Phytoremediation is an effective internal mechanism for the absorption of HMs and their sequestration in various plant tissues.This process enables the removal of HMs from contaminated sites at a faster rate and succeeding harvesting at a lower cost.On the other hand, phytohormones such as SA and JA improve the physiobiochemical processes, increasing plant tolerance to adverse environmental conditions and protecting against various stressors, including HMs.Our findings indicate that SA and JA moderated the deleterious impacts of Ni on physiological parameters.They decreased the levels of CA, MA and His in both populations, thereby modulating plant tolerance to Ni stress.Although the values of phytoremediation indices (TF, BAC, BCF) were greater than one in both populations exposed to Ni (400 μM), a decreasing trend in these values was observed in the presence of SA and JA.Overall, the results revealed that these phytohormones can reduce the phytoremediation Chla mg/g leaves FW = [(12.7 × A 663 ) − (2.69 × A 645 )] × sample weight Chlb mg/g leaves FW = [(22.9× A 645 ) − (4.68 × A 663 )] × sample weight BCF = C root /C solution Vol:.(1234567890)Scientific Reports | (2024) 14:13259 | https://doi.org/10.1038/s41598-024-64336-6

Figure 1 .
Figure 1.Changes in the amounts of chlorophyll a and b in the presence of SA and JA in two populations (NM and M) of A. inflatum stressed with various doses of Ni.All values in the figure are presented as mean ± SE (n = 3).
, treatment with SA and JA in plants in the presence of 400 μM Ni resulted in a reduction in the potential of Ni transport to the aerial parts of the plant (TF value) in both populations.Therefore, the decreasing trend of TF values was shown in 400 μM Ni + 200 μM SA treatment [M population (0.899) and NM population (0.865)], and 400 μM Ni + 10 μM JA treatment [M population (0.73) and NM population (1.084)], in comparison to the plants under Ni (400 μM) stress [M population (1.671) and NM population (1.56)].Similarly, simultaneous treatment of SA and JA in plants under 400 μM Ni resulted in the minimum Ni transport from the root-to-shoot of M and NM population plants (TF < 1).The minimum TF values were detected in 400 μM Ni + 200 μM SA + 10 μM JA treatment in the NM population (0.385), and 400 μM Ni + 50 μM SA + 5 μM JA treatment in the M population (0.476).Our finding indicates that SA and JA probably play an important role in altering the expression of genes involved in the absorption of Ni by the root, transfer to the shoots and its sequestration in the intracellular organelles of both A. inflatum populations by triggering signaling cascades.These phytohormones prevent Ni transpotr and accumulation in shoots by stimulating the decrease of expression of transporter genes involved in Ni absorption and also reducing the content of Ni chelators including His, organic acids and NA 11,25,61 .Similar to our results, Kazemi et al. (2010) informed that the exogenous use of SA in B. napus L. exposed to Ni toxicity caused a discount of Ni accumulation in the shoot.
al. (2016) demonstrated a significant rise of MA content in xylem sap in Ni-stressed B. juncea and Mesembryanthemum crystallinum.While CA content augmented in the Vol.:(0123456789)Scientific Reports | (2024) 14:13259 | https://doi.org/10.1038/s41598-024-64336-6www.nature.com/scientificreports/xylem sap of B. juncea alone led to the accumulation of a greater Ni amount than M. crystallinum 75 .According to our results, in the presence of a high dose of Ni, greater amounts of Ni were accumulated in the shoots than in the roots of both populations of A. inflatum.These outcomes recommend that CA and MA may play a role in long-distance Ni transport and can also perform as intracellular chelators capable of binding Ni in the cytosol or subcellular parts, hence preventing the harmful activity of free HM ions 75 .In contrast to our results, Pietrini et al. (2015) revealed that at higher Ni doses (150 μM), both oxalic and CA could not prevent the deleterious effects on Amaranthus plants at the physiology-biochemical points initiated by this metal.Although in Ni-exposed plants, the content of CA in plant leaves increased relative to the control, with the maximum amount against 25 μM NiCl 2 , in plant roots, the high amount of CA was observed only at 150 μM NiCl 2 .Also, the MA content has not changed in the root and shoot of Amaranthus plants exposed to Ni stress, indicating that this OA in this species may play a minor role as a metal chelator 76 .Dresler et al. (2014) found that in maize seedlings exposed to 50 and 100 μM Cd and Cu stress, CA and MA (at 100 μM Cd) perhaps have a role in Cd transport and sequestration in old leaves, whereas MA is involved in the detoxification of Cu at 50 μM dose.This indicates that CA is one of the Cd chelators involved in the translocation of Cd to older parts of the plant 77 .A reducing trend in MA and CA contents in the existence of SA or JA at 400 μM Ni was observed in most shoots and roots of the M and NM plants against 400 mM Ni in comparison plants stressed to 400 μM only.

Figure 2 .
Figure 2. Changes in CA contents in the presence of SA and JA in two parts of populations (NM and M) of A. inflatum plants stressed with various doses of Ni.All values in the figure are presented as mean ± SE (n = 3).

Figure 3 .
Figure 3. Changes in MA contents in the presence of SA and JA in two parts of populations (NM and M) of A. inflatum plants stressed with various doses of Ni.All values in the figure are presented as mean ± SE (n = 3).
. According to Dalir, and Khoshgoftarmanesh's hypothesis, Ni absorption in the presence of His may occur through two pathways: (I) Ni absorption action by the root may be individually from His absorption, however, the existence of free His around roots indirectly induces Ni absorption by chelating with Ni inside the apoplastic or symplastic spaces of the root.(II) Ni(His) ligands are directly absorbed by the root, and thus, His may enhance Ni absorption through specific His-or Ni(His)-transporters 30 .Similarly, Ali et al. (2009) indicated the increasing concentration of His, serine, and cysteine in the xylem sap of B. napus cultivars exposed to various doses of Ni.In that research, the higher amount of His was attributed to the decontamination of Ni with increased binding to His, serine, and cysteine, and thus indicated Ni tolerance in B. napus84 .Another study demonstrated an increase in the amount of His in the roots and leaf rosettes of Matricaria chamomilla against Ni stress, with a fourfold increase in plant roots at 120 μM Ni85 .In addition, it was found that more His accumulated in Ni hyperaccumulator Alyssum lesbiacum than in non-accumulator Alyssum montanum under Ni stress condition20 .Furthermore,Callahan  et al. (2007) reported a rise in the amount of His and nicotianamine in response to higher Ni concentrations in Thlaspi caerulescens as a serpentine population86 .

Figure 4 .
Figure 4. Changes in His contents in the presence of SA and JA in two parts of populations (NM and M) of A. inflatum plants stressed with various doses of Ni.All values in the figure are presented as mean ± SE (n = 3).

Figure 5 .
Figure 5. Biplot diagram illustrating the principal component analysis (PCA) for the variables studied in the M population.Treatments are depicted in black, while the studied variables are represented by arrows.ShootDW: shoot dry weight; RootDW: root dry weight; R_S ratio: ratio of root/shoot dry weight; Chl a: chlorophyll a; Chl_b: chlorophyll b; S_Ni: shoot Ni content; R_Ni: root Ni content; Shoot_His: shoot histidine content; Root_ His: root histidine content; Shoot_Cit: shoot citric acid content; Root_Cit: root citric acid content; Shoot_Malat: shoot malic acid content; Root_Malat: root malic acid content; BCF: biological concentration factor; BAC: Bioaccumulation coefficient; TF: translocation factor.

Figure 7 .
Figure 7.The biplot diagram displays the outcomes of Principal Component Analysis (PCA) for the studied variables across both populations of M and NM.Treatments are depicted in black, while the studied variables are represented by arrows.ShootDW: shoot dry weight; RootDW: root dry weight; R_S ratio: ratio of root/shoot dry weight ;Chl a: chlorophyll a; Chl_b: chlorophyll b; S_Ni: shoot Ni content; R_Ni: root Ni content; Shoot_His: shoot histidine content; Root_His: root histidine content; Shoot_Cit: shoot citric acid content; Root_Cit: root citric acid content; Shoot_Malat: shoot malic acid content; Root_Malat: root malic acid content; BCF: biological concentration factor; BAC: Bioaccumulation coefficient; TF: translocation factor.

Table 1 .
Changes in DW the presence of SA and JA in two parts of populations (NM and M) of A. inflatum plants stressed with various doses of Ni.All values in the table are presented as mean ± SE (n = 3).

M plant species NM plant species Shoot dry weight (g) Root dry weight (g) Root/shoot ratio (%) Shoot dry weight (g) Root dry weight (g) Root/shoot ratio (%)
def 0.056 ± 0.003mno18.3± 2.29 p-w 0.177 ± 0.005 o-s 0.049 ± 0.003 qrs 27.6 ± 2.05 c-g 10 0.386 ± 0.055 a 0.072 ± 0.002 def 18.9 ± 2.81 k-t 0.174 ± 0.004 o-t 0.035 ± 0.002 z-b 19.8 ± 1.32 k-s 200 0 0.317 ± 0.011 c-h 0.080 ± 0.002 ab 25.3 ± 1.44 f-i 0.192 ± 0.004 o-s 0.063 ± 0.002 jk 32.9 ± 0.95 ab 5 0.309 ± 0.048 d-h 0.080 ± 0.005 ab 26.2 ± 4.73 d-i 0.213 ± 0.006 o-s 0.057 ± 0.004 mno 26.5 ± 2.45 d-h 10 0.363 ± 0.043 a 0.084 ± 0.001 a 23.3 ± 2.37 g-n 0.194 ± 0.005 o-s 0.043 ± 0.003 t-w 22.1 ± 0.65 j-q . Furthermore, matching outcomes were detected in mustard plants in which Ni stress at concentrations of 50, 100, and 150 μM meaningfully decreased the amounts of chlorophyll by 22.15, 30.95, and 40.64%, respectively.In this study, the application of exogenous SA to Ni-stressed plants promoted chlorophyll amounts by 34.2%, 47.2% and 41.8% in the (50, 100 and 150 μM) Ni + SA treatments with respect to plants treated with Ni alone . The maximum Ni concentrations in the shoots of M and NM populations under Ni (400 μM) stresses were 1392 and 1382 μg/g DW, respectively, which was 88.2-and 71.6 times more than the aerial parts of untreated plants.The use of SA and JA alone and/or in combination in plants under Ni stress led to various accumulation patterns of Ni in the root and shoot of M and NM populations.Therefore, Ni uptake in root of the M population treated with the highest amounts of Ni, SA, and JA, i.e. 400 μM Ni + 200 μM SA and also 400 μM Ni + 10 μM JA was 12.6% and 38.4% respectively, with respect to plants treated with 400 μM Ni alone.However, in root of the NM population, the uptake of Ni at the highest dose of Ni and SA increased (6.24%), while it decreased (approximately 7.33%) against the highest dose of Ni and JA relative to the treated plants with 400 μM Ni alone.Furthermore, in M population plants treated with of 400 μM Ni + 200 μM SA + 10 μM JA, a higher Ni uptake (38.2%) was recorded in the root.While in the Ni-treated NM population root, the interaction of JA and SA led to the reduction in Ni uptake with the minimum amount at the treatments of 100 μM Ni + 50 μM SA + 5 μM JA and then at 400 μM Ni + 50 μM SA + 5 μM JA comparison with Ni treatments alone.Similar to our results, in soybean plants compared with leaves, a higher amount of Ni accumulated in plant roots against Ni stress with or without JA-priming.JA-priming in this plant eventuated in a lesser Ni accumulation in different parts of the plant, thereby lessening the damaging impacts of Ni and 59gmenting the growth yield of soybean.This was attributed to the stimulation role of JA in the production of OAs, for example, citrate and malate in root's exudates or thiol compounds to Ni sequestration59.On the other hand,Khalid et al. (2023)revealed that foliar 100 μM SA spray led to a decline in mercury (Hg) accumulation in the aerial parts, root, and fruit of Capsicum annum L. exposed to various doses of Hg (0, 50, 100 and 150 μM).

Table 2 .
Changes in Ni levels and phytoremediation factors (BCF, BAC and TF) in the presence of SA and JA in the M population of A. inflatum stressed with various doses of Ni.All values in the table are presented as mean ± SE (n = 3).
64kewise, this decreasing trend was detected with the rise of Ni dose to 400 μM in BAC value with 9.3% and 14.6% in M and NM populations, respectively, in comparison to the groups treated with Ni (100 μM) alone.In addition, the TF value was less than 1 (TF < 1) in both populations at 100 µM Ni, reaching higher than 1 when the Ni dose increased to 400 µM [M population (1.671), NM population (1.56)].Since the BCF, TF, and BAC values for Ni are greater than 1, two populations of A. inflatum could be used as Ni-hyperaccumulator plants for phytoremediation of Ni-contaminated soil.Similarly, it was found that the accumulation and translocation of Ni in various parts of Alyssoides utriculata found that BCF and TF are intensely more than 1 and this plant is a suitable candidate for Ni phytoremediation from serpentine soils64.Also, in an investigation performed bySajad et al. (2020)to identify plants capable of phytoremediation of Ni from sixty-one sites in Pakistan, it was found that most plant species did not belong to the Ni hyperaccumulators group.Founded on the results of estimating the Ni doses in the soil, root and shoot, also analyzing the values of BCF, TF, and BAC, most of the species have the ability to Ni phytoremediation, for example, Xanthium strumarium (BCF: 89.97) suggested to phytostabilization and Bryophyllum daigremontianum (with TF: 2.37 and BAC: 198.11) suggested to phytoextraction Vol.:(0123456789) Scientific Reports | (2024) 14:13259 | https://doi.org/10.1038/s41598-024-64336-6www.nature.com/scientificreports/ in Tables2 and 3, which can help to investigate the phytoremediation ability of two populations by explaining the characteristics of Ni accumulation and transport behaviors in them.The values of BCF and BAC of both populations under different Ni doses were higher than one (BAC > 1, BCF > 1), while the TF value was higher than 1 in high Ni concentration.The plants' roots had the highest BCF value at 100 μM Ni concentration [M population (6.025), NM population (5.202)]; however, a trend of decrease in BCF value was observed in M (2.08) and NM (2.217) populations of A. inflatum with the increase of Ni doses to 400 μM.

Table 3 .
Changes in Ni levels and phytoremediation factors (BCF, BAC and TF) in the presence of SA and JA in the NM population of A. inflatum stressed with various doses of Ni.All values in the table are presented as mean ± SE (n = 3).
comparison with Ni (100 μM) treatment [M population(3.837),NM population (4.049)].Also, the highest BFC value was recorded in the M population of A. inflatum in simultaneous treatments of 200 μM SA + 10 μM JA exposed to 100 μM (8.952) and 400 μM (3.364) of Ni in comparison with plants against Ni [100 (6.025) and 400 (2.08) μM] only.Unlike the roots, the BAC value displayed a decreasing trend in these treatments.Nevertheless, all these values are higher than one.Similarly, in the NM population treated with 400 μM Ni + 200 μM SA + 10 μM JA, the BFC value exhibited a slight increase, while in this treatment, the BAC value (0.864) decreased significantly with 74.9% compared to the plants under 400 μM Ni stress